The present invention relates to cyanine fluorescent dyes which are functionalized with silanes, their synthesis and their use in the manufacture of fluorescent solid supports, for example fluorescent surfaces or (nano)particles, in the bioconjugation and fluorescent labelling of biomolecules, such as for example nucleosides, nucleotides, nucleic acids (DNA, RNA or PNA), antibodies, proteins and peptides, as well as their use for immobilizing biomolecules on solid supports.
The fluorescent labelling technology is widely used in molecular biology, genomics, proteomics, analytical chemistry, since it is suitable to carry out highly sensitive and specifc tests, efficiently competing with techniques such as radioactive and enzymatic labelling.
DNA probes labelled with fluorescent moieties were shown to be valuable reagents for the separation and analysis of molecules. Specific applications of such fluorescent probes include for example automatic sequencing and DNA mapping; the identification of the concentration of a chemical species capable of binding a second chemical species (for example DNA hybridisation reactions in techniques such as real time PCR, in situ hybridization and molecular recognition with molecular beacons); the localization of biomolecules within cells, tissues or insoluble supports by techniques such as fluorescent staining.
Also fluorescent labelled proteins are very powerful analytical tools which are employed in techniques such as fluorescence microscopy, fluorescent immunoassays, protein chips, cytofluorimetry and laser induced fluorescence capillary electrophoresis.
Among fluorescent dyes, cyanines are widely used as biomolecule labels in several bioanalytical techniques, thanks to their physico-chemical properties such as the high extintion coefficient, the high quantum yield, the independence from pH, the low molecular weight and the possibility to carry out multiple assays with a plurality of fluorophores emitting at different wavelengths. The cyanines are also suitable for use as quenchers if their chemical structure contains suitable groups such as nitro groups.
To be useful as a fluorescent label or quencher in bioconjugations, the cyanine should be provided with a suitable functionalized linker arm having a reactive group.
The research in this filed has therefore focused in the study of innovative functionalized arms, since the chemistry and the behaviour of such linker arms may remarkably affect the fluorescence of the whole molecule as well as other chemical and physical properties such as hydrophobicity/hydrophilicity, aggregation and fluorescence quenching due to intramolecular interactions.
In general, the fluorescent dye molecule may contain a plurality of functionalized linker arms, which are preferably different one from each other in order to avoid the problem of cross-linking between identical molecules, of reticulation, of unwanted reactions or of purification.
There are examples in the literature wherein fluorescent molecules belonging to the class of rhodamines and fluoresceines, as well as luminescent ruthenium complexes, are conjugated to organosilane compounds by coupling reactions that require the use of reactive esters, which are synthesized on the organosilane reagent or on the fluorophore.
However, the use of active esters and generally of active carboxylic groups has remarkable drawbacks, such as the poor stability over time and the difficulty of the synthesis. As a matter of fact, active esters are poorly stable molecules under non anhydrous conditions and are therefore very difficult to store. They are prone to degrade over time by hydrolization and the percentage of active product in a package decreases over time. Moreover, due to their poor stability, it is almost impossible to further store the unused product immediately after opening the package. In addition, the need to work always under perfectly anhydrous conditions makes the synthesis of such compounds particularly difficult and expensive, in that the purification must be performed with the use of anhydrous eluents, especially when the synthesis is conducted on an industrial scale rather than on a lab scale.
International patent application WO2007021946, pages 17-18, discloses the synthesis of cyanines containing a silane functional group by reacting a carboxyl functional group with aminopropyl triethoxysilane in anhydrous pyridine under nitrogen flux for several hours. The final product is treated in anhydrous ether before being purified by chromatography.
The drawbacks of this procedure are the high costs, the complexity and, above all, the need to employ anhydrous solvents. Moreover, the amide bond formed between the cyanine carboxylic acid and the aminopropyl triethoxysilane, or other amine-derivative of silane, is not stable. As a matter of fact, such an amide bond may undergo hydrolization in the aqueous acidic or alkaline environment formed by the conditions required for immobilization of fluorescent derivatives on solid supports, thereby leading again to the formation of the cyanine carboxylic acid and the amino-derivative of silane according to the following scheme:

Similarly, the amide bonds of silane-functionalized cyanines formed by reacting an active ester of a cyanine, such as for example a succinimidyl ester, with an amino-derivative of a silane, may undergo hydrolization.